Antibodies play an important role in clinical applications as well as diagnostic assays. Administration of antibodies specific to host pathogens is an attractive approach to establish protective immunity, employed in passive vaccination or in therapy. Antibodies are often used as vehicles that direct a linked moiety, e.g., an active agent or a toxin, to specific sites in the body, where the antigens recognized by the antibodies are expressed. Labelled antibodies can be used for in vivo diagnostics.
The most common application however is the in vitro diagnostic use of antibodies in immunological assays to identify and/or quantify antigens of interest. The most important formats of immunological tests are ELISA (Enyzme linked immunosorbent assay), RIA (Radioimmunosorbent assay) or ELISPOT (Enzyme linked immunospot), as well as flow cytometric tests such as FACS(Fluorescence activated cell sorting) or blots, e.g., Western blot or dot blot. Antibodies are also used in microscopic applications, such as histology, immunofluorescence or electron microscopy. Antibodies are also used in biotechnological applications, e.g., for purification of the respective antigens.
Typically, antibodies of the Immunoglobulin G (IgG) isotype are used is these applications, although antibodies of other isotypes, e.g., Immunoglobulin M (IgM), are also employed. Polyclonal antibodies are normally generated by immunization of animals, such as mice, rats, rabbits etc., and isolation of antibodies from the serum, following standard protocols. Generation of monoclonal antibodies, which only have a single antigenic specificity, by fusion of antibody-producing lymphocytes from immunized animals with myeloma cells is also well established.
However, use of such antibodies in practice also raises a number of difficulties.
Rheumatoid factor (RF) and human anti-mouse IgG antibodies (HAMA) are probably the most well known causes of false positive or false negative reactions in immunological assays (Boscato L M, Stuart M C, Clin Chem 34, 27-33, 1988). RF is an auto-antibody that reacts with the Fc part of IgG. The disease most often associated with RF is rheumatoid arthritis, but RF can be found in serum from patients with many other diseases and also in 3-5% of healthy blood donors (Johnson P M, Faulk W P, Clin Immunol Immunopathol 6, 414-430, 1976). Production of HAMA is mainly the result of therapeutic approaches with mouse monoclonal antibodies, but HAMA may also be found in serum from patients who have not been treated with antibodies. RF or HAMA may react with both the capture antibody and the detection antibody in a sandwich assay, thereby mimicking antigen activity. A reaction with the detection antibody results in formation of an immune complex which may influence the activity of the detection antibody. HAMA may also react with the antigen-binding region of the detection antibody, thereby impairing or inhibiting antigen binding. The problem of RF and HAMA interference will increase as the sensitivity of the assay increases. Interference by anti-IgG antibodies and antibody-binding substances have been demonstrated in approximately 40% of serum samples from healthy individuals in an immunoradiometric assay (Boscato L, Stuart M, Clin Chem 32, 1491-1495, 1986). RF and HAMA will also give erroneous results in nephelometry and turbidimetry as they change the size of antigen-antibody complex (Chambers R E, et al., Ann Clin Biochem 24, 520-524, 1987).
The prevalence of human anti-mammalian antibodies causing potential interferences in immunological assays varies from 1-80% in the general population (Kricka L J, Clin. Chem. 45, 942-956, 1999).
Furthermore, the IgG antibodies normally used for clinical assays, bound to a solid phase, and antigen-antibody complexes comprising such antibodies, as well as IgM antibodies, can activate the human complement system (Larsson A, Sjoquist J, J Immunol Methods 119, 103-109, 1989). Activated C4 molecules bind to the Fab region of IgG and may interfere with the antigen binding (Campbell R D, et al., Biochem J 189, 6780, 1980). Complement components may also solubilize precipitated immune complexes and prevent soluble immune complexes from precipitating (Baatrup G, et al., Scand J Immunol 23, 397-406, 1986; Miller G W, Nussenzweig V, Proc Natl Acad Sci USA 72, 418-422, 1975).
In clinical laboratories, most analyses are performed on serum samples. A newly obtained serum sample contains active complement, but the activity declines during storage and handling (Whaley, K. Methods in complement for clinical immunologists, Churchill Livingstone, 1985). Accordingly, the complement activity may vary between different patients and also between different samples from the same patient. To avoid activation of the complement cascade, EDTA is often included in tubes used for blood sampling. EDTA prevents complement activation and coagulation by sequestering calcium ions. Most of the standards and controls used have been stored and contain an inactive complement system. This difference in activity between the samples and the standards will cause erroneous results. Complement activation was shown to interfere in an immunometric TSH assay and depressed the TSH values by up to 40% (Kapyaho K, et al., Scand J Clin Lab Invest 49, 211215, 1989).
An additional problem in working with cells or tissue—or with in vivo applications—is the presence of receptors for immunoglobulins. Human FcγRI has a high affinity for monomeric IgG, while FcγRII and FcγRIII mainly bind IgG complexes. There is often some aggregated IgG formed during the purification of IgG or during the labeling procedures that will increase the binding to FcγRII and FcγRIII receptors. Interaction with Fc receptors may cause an increased background staining in immunological tissue analysis. When working with living cells, the interaction with Fc receptors may cause cell activation and changes in the expression of surface proteins. For example, it has been shown that IgG antibodies used in flow cytometry form immune complexes that cause platelet activation and changes in the expression of the GpIlb-IIIa receptor (Lindahl T L, et al., Thromb Haemost 68, 221-225, 1992; Rubinstein E, et al., Br J Haematol 78, 80-86, 1991). Immune complexes containing IgG may also stimulate the production of cytokines (van de Winkel J G, Capel P J, Immunol Today 14, 215-221, 1993).
Similarly, Staphylococcal protein A and Streptococcal protein G are Fc-binding bacterial proteins which are widely used for their ability to bind to IgG. Bacteria of the Staphylococcus aureus Cowan 1 strain and group C Streptococcus sp. are used as immunoadsorbent for IgG. Staphylococci and Streptococci are often found in bacterial samples. When present, they may bind detection antibodies with specificities for other bacteria and cause erroneous results. There are also other bacteria (e.g. Peptostreptococcus magnus, Streptococcus suis and Actinobacillus actinomycetemcomitans) with Ig-binding capabilities (Engstrom P E, et al., J Clin Periodontol 20, 746-751, 1993; Benkirane R, et al., FEMS Immunol Med Microbial 20, 121-127, 1998; Kastern W, et al., Infect Immun 58, 1217-1222, 1990).
One approach to avoid the above mentioned problems, e.g. of cross-reactivity and complement activation, is the use of antibody fragments instead of complete antibodies. For example, IgG antibodies can be enzymatically cut by papain into two Fab fragments (fragment antigen binding) and one Fc fragment (fragment cristallizable). IgG antibodies contain two light and two heavy chains. The light chains have a molecular mass of about 25 kDa and the heavy chains of about 50 kDa. The heavy chain has one variable (V) region and three constant (C) regions. The light chain is composed of one variable and one constant domain. IgG is bivalent, i.e. it has two antigen binding sites. These are composed of the variable domains of both heavy and light chains. The hinge region between the two “arms” of the antibody and the “stem” region gives flexibility to the molecule. Fab fragments thus carry one antigen binding site (they are monovalent).
Recombinant production of Fab fragments is possible. In a preferred form, light and heavy chain domains are formed by a single peptide chain, which can be recombinantly generated (scFv, single chain fragment variable). Libraries of scFv, in particular as phage display libraries, are available in the art, which facilitate generation of recombinant antibodies or scFv specific for a given antigen.
One major limitation of scFv or Fab molecules, however, is their monovalent format, impairing the affinity of these molecules and, thereby, their applicability for therapeutic and diagnostic applications. Utilizing short linker sequences for the construction of scFv, formation of scFv dimers can be favored (Holliger P, et al., Pro. Natl Acad Sci USA 90, 6444, 1993). However, such dimers (‘diabodies’) are formed by noncovalent association and may dissociate. Alternatively, bivalent Fab2 fragments can be used, which still contain the hinge region, wherein the two heavy chains are connected by a disulfide linkage. Fab2 can be produced by enzymatic digestion of antibodies with pepsin.
However, especially for diagnostic applications, another major limitation of scFv, Fab and Fab2 molecules is the lacking Fc region. As a result, many of the advantages offered by antibodies for immunological detection procedures, such as recognition by secondary antibodies to the Fc region, cannot be realized.
The person skilled in the art is therefore faced with the need of generating improved antibodies that avoid or minimize the above mentioned problems in the use of complete IgG antibodies, e.g. of cross-reactivity and complement activation that can lead to false-positive and -negative results in diagnostic assays, but further do not show the disadvantages brought about by use of scFv, Fab and Fab2 molecules.